U.S. patent application number 14/370114 was filed with the patent office on 2015-04-23 for polypeptides inhibiting neovascularization and uses thereof.
The applicant listed for this patent is SHANGHAI FIRST PEOPLE'S HOSPITAL. Invention is credited to Xun Xu, Ying Zheng.
Application Number | 20150111827 14/370114 |
Document ID | / |
Family ID | 48167135 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150111827 |
Kind Code |
A1 |
Xu; Xun ; et al. |
April 23, 2015 |
POLYPEPTIDES INHIBITING NEOVASCULARIZATION AND USES THEREOF
Abstract
Provided is a polypeptide having angiogenesis inhibiting
activity. The polypeptide is derived from Placenta Growth Factor-1.
Also provided are a derivative polypeptide of the polypeptide, a
preparation method for polypeptide, and a pharmaceutical
composition containing the polypeptide.
Inventors: |
Xu; Xun; (Shanghai, CN)
; Zheng; Ying; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHANGHAI FIRST PEOPLE'S HOSPITAL |
Shanghai |
|
CN |
|
|
Family ID: |
48167135 |
Appl. No.: |
14/370114 |
Filed: |
October 29, 2012 |
PCT Filed: |
October 29, 2012 |
PCT NO: |
PCT/CN2012/083671 |
371 Date: |
July 1, 2014 |
Current U.S.
Class: |
514/13.3 ;
530/324; 536/23.5 |
Current CPC
Class: |
C07K 14/515 20130101;
A61K 38/1866 20130101; A61P 9/00 20180101; A61P 35/00 20180101;
A61K 9/0048 20130101; A61P 17/00 20180101; A61P 1/00 20180101; A61P
29/00 20180101; A61P 17/06 20180101; A61P 27/02 20180101; A61K
38/1891 20130101; A61P 15/08 20180101; C07K 14/4703 20130101; A61P
7/02 20180101; A61P 1/04 20180101; A61P 9/10 20180101; A61P 19/02
20180101 |
Class at
Publication: |
514/13.3 ;
530/324; 536/23.5 |
International
Class: |
C07K 14/47 20060101
C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 2011 |
CN |
201110335251.1 |
Claims
1. A polypeptide represented by the following formula I, or a
pharmaceutically acceptable salt thereof, TABLE-US-00006
[Xaa0]-[Xaa1]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6]-
[Xaa7]-[Xaa8]-[Xaa9]-[Xaa10]-[Xaa11]-[Xaa12]-
[Xaa13]-[Xaa14]-[Xaa15]-[Xaa16]-[Xaa17]-[Xaa18]-
[Xaa19]-[Xaa20]-[Xaa21]-[Xaa22]-[Xaa23]-[Xaa24]-
[Xaa25]-[Xaa26]-[Xaa27]-[Xaa28] (I)
wherein, Xaa0 is absent, or selected from Lys, Glu, Pro-Ile-Lys, or
Ile-Lys; Xaa1 is selected from the group consisting of Thr and Ser;
Xaa2 is selected from the group consisting of Ala, Val, Leu and
Ile; Xaa3 is selected from the group consisting of Asn, Gln, His,
Lys or Arg; Xaa4 is selected from the group consisting of Val, Ile,
Leu, Met, Phe and Ala; Xaa5 is selected from the group consisting
of Thr and Ser; Xaa6 is selected from the group consisting of Met,
Leu, Phe, and Ile; Xaa7 is selected from the group consisting of
Gln and Asn; Xaa8 is selected from the group consisting of Leu,
Ile, Val, Met, Ala and Phe; Xaa9 is selected from the group
consisting of Leu, Ile, Val, Met, Ala and Phe; Xaa10 is selected
from the group consisting of Lys, Arg, Gln and Asn; Xaa11 is
selected from the group consisting of Ile, Leu, Val, Met, Ala and
Phe; Xaa12 is selected from the group consisting of Arg, Pro, Lys,
Gln and Asn; Xaa13 is selected from the group consisting of Ser and
Thr; Xaa14 is selected from the group consisting of Gly, Pro, and
Ala; Xaa15 is selected from the group consisting of Asp, Glu; Xaa16
is selected from the group consisting of Arg, Lys, Gln and Asn;
Xaa17 is selected from the group consisting of Pro and Ala; Xaa18
is selected from the group consisting of Ser and Thr; Xaa19 is
selected from the group consisting of Tyr, Trp, Phe, Thr and Ser;
Xaa20 is selected from the group consisting of Val, Ile, Leu, Met,
Phe and Ala; Xaa21 is selected from the group consisting of Glu and
Arg; Xaa22 is selected from the group consisting of Leu and Ser;
Xaa23 is selected from the group consisting of Thr and Arg; Xaa24
is selected from the group consisting of Phe and Ser; Xaa25 is
selected from the group consisting of Ser and Arg; Xaa26 is
selected from the group consisting of Gln and Ser; Xaa27 is
selected from the group consisting of His and Arg; Xaa28 is absent,
or a peptide segment consisting of 1-3 amino acids; and said
polypeptide exhibits an activity of inhibiting angiogenesis.
2. The polypeptide of claim 1, wherein Xaa28 is a peptide segment
consisting of 3 amino acids.
3. The polypeptide of claim 1, wherein Xaa0 is Lys.
4. The polypeptide of claim 1, wherein said polypeptide is selected
from the group consisting of: (a) a polypeptide having the amino
acid sequence represented by SEQ ID NO:1; (b) a polypeptide which
is derived from the polypeptide of (a) by substitution, deletion,
or addition of 1-5 amino acids in the amino acid sequence of SEQ ID
NO: 1 and which has the activity of inhibiting angiogenesis;
wherein, the homology between the derived polypeptide and SEQ ID
NO.: 1 is .gtoreq.95%.
5. An isolated nucleic acid molecule encoding the polypeptide of
claim 1.
6. A pharmaceutical composition comprising: (a) the polypeptide or
a pharmaceutically acceptable salt thereof of claim 1; and (b) a
pharmaceutically acceptable carrier or excipient.
7. The pharmaceutical composition of claim 6, wherein the dosage
form of the composition is injection solution, eyedrop, ophthalmic
gel or eye ointment.
8. (canceled)
9. (canceled)
10. A method for inhibiting angiogenesis in a mammal, comprising
the step of administering a subject in need thereof with the
polypeptide or a pharmaceutically acceptable salt thereof of the
present invention.
11. The method of claim 10, wherein, said angiogenesis is selected
from the group consisting of neovascular eye diseases, tumor,
ischemic heart disease, non-inflammatory myocardiopathy, coronary
sclerosis, arteriosclerosis obliterans, artery embolism, artery
thrombus, Berger's disease, chronic inflammation, inflammatory
intestinal diseases, ulcer, rheumatic arthritis, scleroderma,
psoriasis, infertility and sarcoma-like diseases.
Description
TECHNICAL FIELD
[0001] The present invention relates to biomedicine. In particular,
the present invention relates to a novel small peptide inhibiting
angiogenesis, and said small peptide is a polypeptide derived from
Placenta Growth Factor (PLGF). The present invention also relates
to a method for preparing the polypeptide, uses thereof, and a
pharmaceutical composition comprising the polypeptide.
TECHNICAL BACKGROUND
[0002] Angiogenesis involves extremely complicated courses
including expansion of existing vessels, increase in vascular
permeability, degradation of perivescular stroma, activation,
proliferation and migration of endothelial cells, and formation of
new capillary-like lumina.
[0003] About 2/3 of diseases causing blindness are associated with
pathological angiogenesis in eyes, for example, corneal
angiogenesis induced by simplex herpetic stromal keratitis,
choroidal angiogenesis in age-related macular degeneration, and
retinal angiogenesis in diabetic retinopathy or retinopathy of
premature infant. At present, laser photocoagulation, photodynamic
therapy (PDT), and thermal transpupillary therapy (TTT) etc. are
conventionally used for clinically treating the ocular pathological
angiogenesis. However, these treatments tend to destroy local
tissues, and the long-term efficacy thereof is still
unsatisfactory. Therefore, in recent years, people kept trying to
develop more effective methods for treating ocular pathological
angiogenesis.
[0004] When developing effective inhibitors of angiogenesis, the
specificity of the ocular drugs should be sufficiently
considered.
[0005] Firstly, there are many anatomical and functional barriers
in eyes. Systemic administration usually cannot result in a
topically sufficient drug concentration in ocular tissue due to the
blood-aqueous humor barrier and blood-retina barrier.
Theoretically, in topical administration, such as injection in
vitreous cavity, it is difficult for any macromolecule larger than
76.5 kDa to penetrate the retina and act on the retinal and
choroidal angiogenesis. When administrated on ocular surface, the
drugs have to successively penetrate lipophilic the corneal
epithelial cells as well as the hydrophilic corneal stroma, which
are the tightly connected. Thus, merely the medications that have
appropriate lipophilicity, a low molecular weight or capability to
bind with the transporters (e.g., amino acid transporters,
oligopeptide transporters, etc.) in ocular surface tissues can
reach the anterior chamber and function effectively.
[0006] Secondly, the solubility of the drugs in the hydrophilic
tears, aqueous humor, and vitreous humor is positively correlated
to their effects.
[0007] Thirdly, for the above major reasons, the bioavailability of
ocular drugs is very low. To improve it, the administration
concentration of drugs should be increased.
[0008] However, compounds for treating neoplastic angiogenesis
exhibit significant toxicity, so that high dose cannot be used in
either systemic or topical administration. In addition, exogenous
proteins with large molecular weight are also sensitive foreign
substances which may cause immune damages to eye tissues such as
uveal.
[0009] Fourthly, currently a series of relatively safe endogenous
inhibitors of angiogenesis, such as angiostatin consisting of
plasminogen Kringle domains 1-4, have been demonstrated to
significantly inhibit growth of vessel blood-dependent tumor.
However, due to their relative large molecular weight and
complicated spatial conformation, these inhibitors have
disadvantages in preparation such as complicated recombinant
expression and purification processes, residual endotoxin and so
on.
[0010] Because of the constraints caused by the above factors, at
present, only a few medicaments are used for treating ocular
angiogenesis, e.g., recombinant anti-VEGF monoclonal antibody
bevacizumab (Avastin), and the recombinant fragment of anti-human
VEGF monoclonal antibodies ranibizumab (Lucentis), etc. However,
they are expensive, repeated intravitreal administrations are
necessary, and certain risks, such as vascular embolization will be
caused.
[0011] Thus, for preventing and treating neovascular eye diseases,
it is extremely important to seek for the small-molecule inhibitors
with specific biological activity and biocompatibility, which can
penetrate all kinds of barriers in ocular tissue via non-invasive
or minimally invasive administration, thereby enhancing ocular
bioavailability with reduced dosage, reduced side effects either
locally or systemically. Therefore, there is an urgent need in
developing small molecule inhibitors of angiogenesis, which are
safe, effective, and compatible with eyeball tissues.
SUMMARY OF INVENTION
[0012] The purpose of the present invention is to provide a small
molecular polypeptide, and the fragments, analogs, and derivatives
thereof, which are suitable for eyeball tissue, effective and safe
for inhibit angiogenesis.
[0013] Another purpose of the present invention is to provide a
method for preparing said polypeptide and use of said
polypeptide.
[0014] In the first aspect, the present invention provides a
polypeptide represented by the following formula I, or a
pharmaceutically acceptable salt thereof,
TABLE-US-00001 [Xaa0]-[Xaa1]-[Xaa2]-[Xaa3]-[Xaa4]-[Xaa5]-[Xaa6]-
[Xaa7]-[Xaa8]-[Xaa9]-[Xaa10]-[Xaa11]-[Xaa12]-
[Xaa13]-[Xaa14]-[Xaa15]-[Xaa16]-[Xaa17]-
[Xaa18]-[Xaa19]-[Xaa20]-[Xaa21]-[Xaa22]-
[Xaa23]-[Xaa24]-[Xaa25]-[Xaa26]-[Xaa27]- [Xaa28] (I)
wherein,
[0015] Xaa0 is absent, or a peptide segment consisting of 1-3 amino
acids;
[0016] Xaa1 is selected from the group consisting of Thr and
Ser;
[0017] Xaa2 is selected from the group consisting of Ala, Val, Leu
and Ile;
[0018] Xaa3 is selected from the group consisting of Asn, Gln, His,
Lys and Arg;
[0019] Xaa4 is selected from the group consisting of Val, Ile, Leu,
Met, Phe and Ala;
[0020] Xaa5 is selected from the group consisting of Thr and
Ser;
[0021] Xaa6 is selected from the group consisting of Met, Leu, Phe,
or Ile;
[0022] Xaa7 is selected from the group consisting of Gln and
Asn;
[0023] Xaa8 is selected from the group consisting of Leu, Ile, Val,
Met, Ala and Phe;
[0024] Xaa9 is selected from the group consisting of Leu, Ile, Val,
Met, Ala and Phe;
[0025] Xaa10 is selected from the group consisting of Lys, Arg, Gln
and Asn;
[0026] Xaa11 is selected from the group consisting of Ile, Leu,
Val, Met, Ala and Phe;
[0027] Xaa12 is selected from the group consisting of Arg, Pro,
Lys, Gln and Asn;
[0028] Xaa13 is selected from the group consisting of Ser and
Thr;
[0029] Xaa14 is selected from the group consisting of Gly, Pro, and
Ala;
[0030] Xaa15 is selected from the group consisting of Asp, Glu;
[0031] Xaa16 is selected from the group consisting of Arg, Lys, Gln
and Asn;
[0032] Xaa17 is selected from the group consisting of Pro and
Ala;
[0033] Xaa18 is selected from the group consisting of Ser and
Thr;
[0034] Xaa19 is selected from the group consisting of Tyr, Trp,
Phe, Thr and Ser;
[0035] Xaa20 is selected from the group consisting of Val, Ile,
Leu, Met, Phe and Ala;
[0036] Xaa21 is selected from the group consisting of Glu and
Arg;
[0037] Xaa22 is selected from the group consisting of Leu and
Ser;
[0038] Xaa23 is selected from the group consisting of Thr and
Arg;
[0039] Xaa24 is selected from the group consisting of Phe and
Ser;
[0040] Xaa25 is selected from the group consisting of Ser and
Arg;
[0041] Xaa26 is selected from the group consisting of Gln and
Ser;
[0042] Xaa27 is selected from the group consisting of His and
Arg;
[0043] Xaa28 is absent, or a peptide segment consisting of 1-3
amino acids;
[0044] and said polypeptide exhibits an activity of inhibiting
angiogenesis and has a length of 27-33 amino acids.
[0045] In another preferred embodiment, said polypeptide has a
length of 28-31 amino acids.
[0046] In another preferred embodiment, Xaa28 is a peptide segment
consisting of 3 amino acids.
[0047] In another preferred embodiment, Xaa0 is absent, or a
peptide segment consisting of 1, 2 or 3 amino acid(s).
[0048] In another preferred embodiment, Xaa0 is selected from Lys,
Glu, Pro-Ile-Lys, or Ile-Lys.
[0049] In another preferred embodiment, said polypeptide is
selected from the group consisting of:
[0050] (a) a polypeptide having the amino acid sequence represented
by SEQ ID NO:1;
[0051] (b) a polypeptide which is derived from the polypeptide of
(a) by substitution, deletion, or addition of 1-5 amino acids
(preferably 1-3, and more preferably 1-2) in the amino acid
sequence of SEQ ID NO: 1 and which has the activity of inhibiting
angiogenesis.
[0052] In another preferred embodiment, said derived polypeptide
retains .gtoreq.70% activity to inhibit angiogenesis of polypeptide
represented by SEQ ID NO.: 1.
[0053] In another preferred embodiment, the identity between said
derived polypeptide and SEQ ID No.: 1 is .gtoreq.80%, preferably
.gtoreq.90%; and more preferably .gtoreq.95%.
[0054] The present invention further provides a dimer form and a
polymer form of the compound of formula I, which exhibit the
activity of inhibiting angiogenesis.
[0055] In the second aspect, the present invention provides an
isolated nucleic acid molecule encoding the above polypeptide of
the present invention.
[0056] In the third aspect, the present invention provides a
pharmaceutical composition comprising:
[0057] (a) the above polypeptide or a pharmaceutically acceptable
salt thereof of the present invention; and
[0058] (b) a pharmaceutically acceptable carrier or excipient.
[0059] In another preferred embodiment, the composition is in the
form of eyedrop, injection solution (such as injection solution for
periocular or intraocular injection), ophthalmic gel or eye
ointment.
[0060] In another preferred embodiment, the composition is in a
sustained release dosage form.
[0061] In the fourth aspect, the present invention provides a use
of said polypeptide or a pharmaceutically acceptable salt thereof
for preparing a medicament for inhibiting angiogenesis, or
preventing or treating diseases associated with angiogenesis.
[0062] In another preferred embodiment, the disease associated with
angiogenesis is selected from the group consisting of neovascular
eye diseases, tumor, ischemic heart disease, non-inflammatory
myocardiopathy, coronary sclerosis, arteriosclerosis obliterans,
artery embolism, artery thrombus, Berger's disease, chronic
inflammation, inflammatory intestinal diseases, ulcer, rheumatic
arthritis, scleroderma, psoriasis, infertility or sarcoma-like
diseases.
[0063] In another preferred embodiment, the neovascular eye
diseases include diseases involved in choroid, retina, cornea or
iris, including age-related macular degeneration, proliferative
diabetic retinopathy, retinal vessel-blocked diseases, retinopathy
of prematurity, corneal infection, and neovascular glaucoma.
[0064] In the fifth aspect, the present invention provides a method
for inhibiting angiogenesis in mammal, comprising the step of
administering the polypeptide or a pharmaceutically acceptable salt
thereof of the present invention to a subject in need thereof.
[0065] In another preferred embodiment, the subject is a human.
[0066] In another preferred embodiment, the angiogenesis is
associated with neovascular eye diseases.
[0067] It should be understood that in the present invention, the
technical features specifically described above and below (such as
the Examples) can be combined with each other, thereby constituting
a new or preferred technical solution which needs not be described
one by one.
DESCRIPTION OF DRAWINGS
[0068] The following descriptions of drawings are intended to
illustrate the specific embodiments of the present invention, but
not to limit the scope of the present invention, which should be
defined by the claims.
[0069] FIG. 1 shows the purity identification of the Small peptide
ZY3 analyzed by High Performance Liquid Chromatography (HPLC).
[0070] FIG. 2 shows the effect of Small peptide ZY3 on
proliferation of Human Umbilical Vein Endothelial Cells (HUVECs).
Small peptide ZY3 has a significant effect of inhibiting the
proliferation of endothelial cells. Compared with VEGF group, in
groups of VEGF+ZY3 peptide, the proliferation of HUVECs are
significantly inhibited. *P<0.05, **P<0.01. The differences
are statistically significant.
[0071] FIG. 3 shows the effect of Small peptide ZY3 on lumen
formation of Human Umbilical Vein Endothelial Cells (HUVECs). Small
peptide ZY3 exhibits a significant effect of inhibiting the lumen
formation of endothelial cells. FIG. 3a, FIG. 3b, and FIG. 3c show
that ZY3 inhibits the lumen formation of HUVECs. FIG. 3a is the
VEGF group; FIG. 3b is the VEGF+ZY3(160 .mu.M) group; FIG. 3c
indicates that in groups of VEGF with Small peptide ZY3 in
different concentrations, lumen formation of HUVECs is
significantly inhibited. *P<0.05. The differences are
statistically significant.
[0072] FIG. 4 shows the effect of Small peptide ZY3 on angiogenesis
in chick embryo chorioallantoic membrane: Small peptide ZY3
exhibits a significant effect on anti-angiogenesis. FIG. 4a-4c show
the count results of 3-5 subordinate microvessels in the area
within 2.5 mm around the filter paper. FIG. 4a is the PBS group;
FIG. 4b is the ZY3(10 .mu.l 1/piece) group; FIG. 4c is the ZY3(50
.mu.l/piece) group; FIG. 4d shows that, compared with the VEGF
group, in the groups of VEGF+small peptide ZY3 with different
concentrations, the neovascularization of chick embryo
chorioallantoic membrane is significantly inhibited and the
inhibition is concentration-dependent. **P<0.01. The differences
are statistically significant.
[0073] FIG. 5 shows the effect of small peptide ZY3 on pathological
angiogenesis in mouse cornea. Small peptide ZY3 exhibits a
significant inhibition effect on angiogenesis. FIG. 5a-5c show the
neovascularization area on mouse cornea. FIG. 5a is the VEGF group;
FIG. 5b is the ZY3(0.5 .mu.l/granule) group; FIG. 5c is the ZY3(2
.mu.l 1/granule) group; FIG. Sd shows that, compared with the VEGF
group, in the groups of VEGF+ small peptide ZY3 with different
concentrations, the pathological angiogenesis in mouse cornea is
significantly inhibited. **P<0.01. The differences are
statistically significant.
DETAILED DESCRIPTION OF THE INVENTION
[0074] After extensive and intensive studies, the inventors have
firstly prepared a class of small molecular polypeptides derived
from Placental Growth Factor, exhibiting a function of angiogenesis
inhibition and having a molecular weight of less than 5 kD (for
example, just about 3 KD). In particular, by utilizing the method
of bioinformatics, the inventor designed several candidate
sequences based on the homology analysis and analysis on the
biological properties. After synthesizing these sequences via solid
phase synthesis, obtaining small peptide ZY3 with high purity upon
purification and further screening through the model of vessels on
chick embryo chorioallantoic membrane, the model of VEGF induced
HUVECs proliferation and lumen formation, the mouse model of VEGF
induced corneal angiogenesis, and the mouse model of corneal
micro-capsule, the inventors obtained a class of novel, small
molecular polypeptides exhibiting the function of preventing and
treating angiogenesis.
[0075] The molecular weight of small peptides of the present
invention is small, enabling the peptides penetrating through
various ocular tissue barriers. They have good water solubility, so
that they can maintain a relatively high concentration in neutral
tears, aqueous humor and vitreous humor. They are highly safe with
a minor toxicity or side-effect to the biological tissue. The
bioavailability is high through local application in the eye,
thereby reducing either the dose or the systemic toxicity. Based on
the above works, the present invention is completed.
[0076] Placenta Growth Factor
[0077] Placenta Growth Factor (PlGF) is one of the VEGF family.
PlGF was separated and purified by Maglione et.al from the DNA
library of human placenta as early as 1991. PlGF could be detected
in heart, lung, thyroid, skeletal muscles besides in human
placenta. Based on the selective splicing of PlGF genes, 4
different isoforms in molecular size, secretion features, and
receptor affinity could be formed: PlGF-1(PlGF131),
PlGF-2(PlGF152), PlGF-3(PlGF203), and PlGF-4(PlGF224). Two PlGF
monomers form secreted homodimer glycoprotein, and then bind to
their receptors, thereby mediating the following signal
transduction and exerting their biological effects. In addition,
heterodimers could be formed by combining PlGF with VEGF to affect
the signal transduction pathway of VEGF. PlGF can promote
proliferation of endothelial cells, especially microvascular
endothelial cells and it can be used as the chemokine for
endothelial cells growth factor to regulate endothelial cells
growth and stimulate angiogenesis. PlGF can further promote the
migration of monocytes and endothelial cells to increase the
permeability of endothelial cells. Although angiogenesis can also
be induced by VEGF, the new blood vessel induced by PlGF exhibits
normal physiological characteristics without any other abnormality.
The new blood vessel induced by PlGF won't exhibit phenomena, such
as edema, hemangioma and increased permeability due to the VEGF
induced angiogenesis.
[0078] Active Polypeptides
[0079] In the present invention, the terms "the polypeptide(s) of
the present invention", "polypeptide(s) ZY3", "small peptide(s)
ZY3", "short peptide(s) ZY3" and "peptide(s) ZY" are
interchangeably used and refer to a protein or polypeptide having
peptide ZY3 amino acid sequence (TANVTMQLLKIRSGDRPSYVELTFSQH, SEQ
ID NO: 1) and exhibiting an activity of inhibiting angiogenesis. In
addition, said terms comprise the variants of SEQ ID NO: 1 which
exhibit the function of inhibiting angiogenesis. These variations
include, but are not limited to, deletions, insertions and/or
substitutions of 1-5 (typically 1-4, preferably 1-3, more
preferably 1-2, most preferably 1) amino acids, and addition of one
or more (typically less than 5, preferably less than 3, more
preferably less than 2) amino acids at C-terminus and/or
N-terminus. For example, a protein's functions are usually
unchanged when an amino residue is substituted by a similar or
analogous one in the art. Further, the addition of one or several
amino acids at C-terminus and/or N-terminus generally will not
change the structure and function of protein. Furthermore, the
terms also include the polypeptide of the present invention in
monomer and polymer form. The terms also include the linear and
nonlinear polypeptides (such as cyclic peptides).
[0080] The present invention further includes the active fragments,
derivatives and analogs of ZY3 polypeptide. As used herein, the
terms "fragments", "derivatives" and "analogs" refer to the
polypeptides substantially maintaining the function or activity of
inhibiting angiogenesis. The polypeptide fragments, derivatives or
analogs of the present invention may be (i) a polypeptide with one
or more conservative or non-conservative amino acid residues
(preferably the conservative amino acid residues) being
substituted, or (ii) a polypeptide having substituted group(s) in
one or more amino acid residues, or (iii) a polypeptide formed by
fusion of ZY3 polypeptide with another compound (such as the
compound that prolongs the half life of the polypeptide, such as
polyethylene glycol), or (iv) a polypeptide with additional amino
acid sequence fused to said polypeptide sequence, such as fusion
proteins formed by fusion with leader sequence, secretion sequence
or tag sequence, such as 6H is. According to the subject
application, these fragments, derivatives and analogs are within
the scope commonly known by the skilled person.
[0081] A class of preferred active derivatives is the polypeptides
formed by replacing at most 5, preferably at most 3, more
preferably at most 2, most preferably 1 amino acid of the amino
acid sequence represented by formula I with the amino acid having
similar or analogous property. These conservative variant
polypeptides are preferably formed by carrying out the amino acid
replacement according to Table I.
TABLE-US-00002 TABLE I Initial Representative Preferred residue
substitution substitution Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Lys; Arg Gln Asp (D) Glu Glu Cys (C)
Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His
(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe Leu Leu
(L) Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M)
Leu; Phe; Ile Leu Phe (F) Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala
Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y)
Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala Leu
[0082] The present invention also provides the analogues of ZY3
polypeptide. These analogues differ from naturally occurring ZY3
polypeptide in amino acid sequence or modifications that do not
affect the sequence, or by both. Also included are analogues which
include residues other than those naturally occurring L-amino acids
(e.g., D-amino acids) or non-naturally occurring or synthetic amino
acids (e.g., beta- or gamma-amino acids). It is understood that the
polypeptides of the present invention are not limited to the
representative polypeptides listed hereinabove.
[0083] Modifications (which do not normally alter the primary
sequence) include in vivo or in vitro chemical derivation of
polypeptides, e.g., acelylation, or carboxylation. Glycosylation is
also included in modification, e.g., the polypeptides produced by
glycosylation modification during its synthesis and processing or
in the further processing steps. These modifications can be
achieved by exposing the polypeptide to enzymes for glycosylation
(e.g., mammalian glycosylating or deglycosylating enzymes). Also
included are sequences that have phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, phosphothronine, as
well as sequences that have been modified to improve their
resistance to proteolytic degradation or to optimize solubility
properties.
[0084] The polypeptides of the present invention can be used in a
form of pharmaceutically or physiologically acceptable salt derived
from acid or base. Such salts include, but are not limited to, the
salts formed with the following acids: hydrochloric acid,
hydrobromic acid, sulfuric acid, citric acid, tartaric acid,
phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic
acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid,
methanesulfonic acid, ethyl-sulfonic acid, benzene sulfonic acid,
or isethionic acid. Other salts include salts formed with alkali
metals or alkaline earth metals (such as sodium, potassium, calcium
or magnesium), and esters, carbamate or other conventional
"prodrug" forms.
[0085] Encoding Sequences
[0086] The present invention further relates to a polynucleotide
encoding ZY3 polypeptide. A preferred encoding sequence which
encodes ZY3 short peptide as shown in SEQ ID NO.: 1 is
TABLE-US-00003 (SEQ ID NO: 2:
ACGGCCAATGTCACCATGCAGCTCCTAAAGATCCGTTCTGGGGACCGGC
CCTCCTACGTGGAGCTGACGTTCTCTCAGCAC).
[0087] The polynucleotide of the present invention can be in a form
of DNA or RNA. DNA can be the coding strand or the non-coding
strand. The coding sequence encoding the mature polypeptide can be
identical to the coding sequence indicated in SEQ ID NO: 2, or can
be a degenerate variant thereof. As used herein and taking SEQ ID
NO.: 2 as an example, "degenerate variant" refers to a nucleic acid
sequence which encodes the protein having the amino acid sequence
of SEQ ID NO:1, but is different from the corresponding coding
sequence in SEQ ID NO: 2.
[0088] ZY3 full-length nucleotide sequence or a fragment thereof of
the present invention can be obtained via PCR amplification,
recombination method or artificial synthesis. Currently, the DNA
sequence encoding the polypeptide (or a fragment or derivative
thereof) of the present invention can be prepared completely via
chemical synthesis. Then the DNA sequence can be introduced into
various existing DNA molecules (or such as vector) and cells known
in the art.
[0089] The present invention also includes a vector containing the
polynucleotide of the present invention, and a host cell engineered
by the vector or the coding sequence of the ZY polypeptide of the
present invention.
[0090] In another aspect, the present invention further comprises
polyclonal antibodies or monoclonal antibodies specific to ZY3
polypeptide, especially the monoclonal antibodies.
[0091] Preparation Method
[0092] The polypeptide of the present invention can be a
recombinant or synthetic polypeptide. The polypeptide of the
present invention can be a chemically synthesized or recombinant
polypeptide. Accordingly, the polypeptide of the present invention
can be artificially synthesized via a conventional method, or can
be produced via a recombinant method.
[0093] One preferred method is to use liquid phase synthesis
technique or solid phase synthesis technique, such as Boc solid
phase process, Fmoc solid phase process, or combination thereof. By
using the solid phase synthesis, a sample can rapidly be obtained,
and one can select a suitable resin carrier and synthesis system
according to the sequence feature of the target peptide. For
example, the preferred solid phase carrier in Fmoc system can be,
such as Wang resin linked to the C-terminal amino acid of the
peptide, wherein the structure of the Wang resin is polystyrene,
the arm between the resin and the amino acid is 4-alkoxy benzyl
alcohol. The Wang resin is treated with 25%
hexahydropyridine/dimethylfomamide for 20 minutes under room
temperature to remove the Fmoc protective groups. Then the sequence
is extended from the C-terminus to the N-terminus one-by-one
according to the predetermined amino acid sequence. After
synthesis, trifluoroacetic acid containing 4% p-methylphenol is
used to cleave the preinsulin-relevant peptide from the resin and
the protective groups are removed. The resin can be filtered, and
the crude peptide can be obtained via precipitation with ether. The
solution of the resulting product is freeze-dried, gel-filtered,
and purified by reverse phase HPLC to obtain the desired peptide.
When utilizing the Boc system to perform the solid phase synthesis,
preferably the resin is the PAM resin linked to the C-terminal
amino acid of the peptide. The structure of the PAM resin is
polystyrene, and the arm between the resin and the amino acid is
4-hydroxylmethyl phenylacetamide. In the Boc synthesis system, in
the circle of deprotection, neutralization, and coupling,
TFA/dichloromethane (DCM) is used to remove the protective group
Boc, and diisopropylethylamine (DIEA)/dichloromethane is used for
neutralization. After completion of peptide chain condensation,
hydrogen fluoride (HF) containing p-methylphenol (5-10%) is used to
treat the resin for 1 hour at 0.degree. C., then the peptide chain
is cleaved from the resin and the protective groups are removed at
the same time. 50-80% acetic acid (containing a small amount of
mercaptoethanol) is used to extract the peptide. The solution is
freeze-dried, and then further isolated and purified by molecular
screen Sephadex G10 or Tsk-40f. Then the desired peptide is
obtained via high pressure liquid purification. Various coupling
agents and coupling methods known in the peptide chemistry can be
used to couple each amino acid residue. For example,
dicyclohexylcarbodiimide (DCC), hydroxylbenzotriazole (HOBt) or
1,1,3,3-tetramethyluronium Hexafluorophosphate (HBTU) can be used
for direct coupling. The purity and structure of the resulting
short peptide can be verified by reverse phase HPLC and mass
spectrometry.
[0094] In a preferred embodiment, the polypeptide ZY3 of the
present invention is prepared by solid phase method according to
its sequence, purified by high performance liquid chromatography,
thereby obtaining freeze-dried powder of target peptide with high
purity. The powder is stored at -20.degree. C.
[0095] Another method is to produce the polypeptide of the present
invention by a recombination technique. With the conventional
recombinant DNA technique, the polynucleotide of the present
invention can be used to express or produce recombinant ZY3
polypeptides. Generally, the method comprises the following
steps:
[0096] (1) Transforming or transfecting a suitable host cell with a
polynucleotide or variant thereof encoding the ZY3 polypeptide of
the present invention or a recombinant expression vector containing
said polynucleotide;
[0097] (2) Culturing the host cell in a suitable culture
medium;
[0098] (3) Isolating and purifying protein from the culture medium
or cell.
[0099] The recombinant polypeptide may be expressed in cells or on
the cell membrane, or secreted out of the cell. If desired, the
physical, chemical and other properties can be utilized in various
isolation methods to isolate and purify the recombinant protein.
These methods are well-known to those skilled in the art and
include, but are not limited to, conventional renaturation
treatment, treatment by protein precipitant (such as salt
precipitation), centrifugation, cell lysis by osmosis, sonication,
supercentrifugation, molecular sieve chromatography (gel
chromatography), adsorption chromatography, ion exchange
chromatography, high performance liquid chromatography (HPLC), and
any other liquid chromatography, and the combination thereof.
[0100] It is also contemplated to link multiple polypeptides of the
present invention in series due to the short length of the peptide.
After recombinant expression, the expression product is obtained in
a form of polymer. Then the polymer is enzymatically cleaved to
form the desired small peptides.
[0101] Pharmaceutical Composition and Methods of Administration In
another aspect, the present invention further provides a
pharmaceutical composition, comprising (a) a safe and effective
amount of the polypeptide of the present invention or a
pharmaceutically acceptable salt thereof, and (b) a
pharmaceutically acceptable carrier or excipient. The amount of the
polypeptide of the present invention generally is 10 .mu.g to 100
mg per dose, preferably 100-1000 .mu.g per dose.
[0102] For the purpose of the invention, the effective dose is
about 0.01 mg to 50 mg of the polypeptide of the present invention
per kg body weight, preferably 0.05 mg to 10 mg of the polypeptide
of the present invention per kg body weight administered to an
individual. Further, the polypeptide of the present invention can
be used alone, or in combination with the other therapeutic agents
(for example, formulated into the same pharmaceutical
composition).
[0103] The pharmaceutical composition can further comprise a
pharmaceutically acceptable carrier. The term "pharmaceutically
acceptable carrier" refers to the carrier for using in
administering the therapeutic agents. The term refers to such
medical carriers that they themselves do not induce antibody
deleterious to the subject having been administered the
composition, and they do not have excessive toxicity after
administration. These carriers are well known by the skilled person
in the art. The detailed discussion about the pharmaceutically
acceptable excipient can be found in Remington's Pharmaceutical
Sciences (Mack Pub. Co., N.J., 1991). Such carriers include, but
are not limited to, saline, buffer solution, glucose, water,
glycerin, ethanol, adjuvant or the combination thereof.
[0104] The pharmaceutically acceptable carrier in the therapeutic
composition can comprise liquid, such as water, saline, glycerin,
and ethanol. Further, these carriers can contain auxiliary
substance(s), such as wetting agent or emulsifying agent, pH
buffering substance, etc.
[0105] Typically, the therapeutic composition can be formulated
into an injectable formulation, such as a liquid solution or
suspension; or it may be in a solid form that is suitable to be
formulated into a solution or suspension or liquid carrier before
injection.
[0106] Once formulated the composition of the present invention can
be administered via conventional routes which include, but are not
limited to, administering on ocular surface, around the eye,
intra-ocularly, intramuscularly, intravenously, subcutaneously,
intracutaneously or topically. The subject to be prevented or
treated may be an animal, especially a human.
[0107] When the pharmaceutical composition of the present invention
is used in the actual treatment, the dosage form of the
pharmaceutical composition can be varied according to the uses.
Preferably, as an example, the dosage form may include eyedrop,
injection, ophthalmic gel, and eye ointment.
[0108] The pharmaceutical composition can be formulated by mixing,
diluting or dissolving according to the conventional methods. And,
occasionally, suitable medical additives, such as excipients,
disintegrating agents, adhesives, lubricants, diluting agents,
buffering agents, isotonicities, preservatives, wetting agents,
emulsifying agents, dispersing agents, stabilizing agents, and
solubility promoters, may be added. Formulation can be carried out
in a conventional manner according to the dosage form.
[0109] For example, formulation of eyedrop can be prepared as
follows: dissolving short peptide ZY or a pharmaceutically
acceptable salt thereof and the basic substances in sterile water
(surfactant is dissolved in said water), adjusting osmotic pressure
and pH to the physiological level, optionally adding suitable
medical additives, such as preservatives, stabilizing agents,
buffering agents, isotonicities, anti-oxidants and tackifiers, and
then completely dissolving them.
[0110] The pharmaceutical composition of the present invention can
further be administered in a form of sustained release formulation.
For example, the short peptide ZY or salt thereof can be
incorporated into the pill or microcapsule in which a sustained
release polymer is used as carrier, and then the pill or
microcapsule is implanted into the tissue to be treated by
operation. Furthermore, the short peptide ZY3 or salt thereof can
be used by insertion of intra-ocular lens pre-coated with said
drugs. Examples of the slow release polymer include
ethylene-ethylene acetate copolymer, polyhydroxymethylacrylate,
polyacrylamide, polyvinylpyrrolidone, methyl cellulose, polymer of
lactic acid, lactic acid-glycolic acid copolymer, etc. Preferable
examples of the sustained release polymer include the biodegradable
polymers, such as polymer of lactic acid, and lactic acid-glycolic
acid copolymer.
[0111] When the pharmaceutical composition of the present invention
is used in the actual treatment, the dose of the short peptide ZY3
or a pharmaceutically acceptable salt thereof, as an active
ingredient, can be suitably determined according to the body
weight, age, sex, symptom of each patient. For example, when
topically dropping in the eye, the concentration of the active
ingredient generally is 0.1-10 wt %, preferably 1-5 wt %, and the
composition can be administrated for 2-6 times per day with 1-2
drops each time.
INDUSTRIAL APPLICABILITY
[0112] The pharmaceutical composition containing the peptide of the
present invention or a pharmaceutically acceptable salt thereof as
an active ingredient exhibits significant inhibition activity on
angiogenesis. As verified by animal tests, the polypeptide of the
present invention not only can inhibit angiogenesis in chick embryo
chorioallantoic membrane, but also can inhibit the proliferation,
migration, chemotaxis and lumen formation of HUVEC and the retinal
angiogenesis in the hypoxia-induced mice.
[0113] The main advantages of the present invention include:
[0114] (a) The polypeptide ZY3 of the present invention has small
molecular weight, so that it can penetrate ocular tissue
barrier.
[0115] (b) The polypeptide of the present invention has good water
solubility, so that it can maintain relatively high concentration
in neutral tears, aqueous humor and vitreous humor.
[0116] (c) The polypeptide of the present invention has high safety
with less toxicity to the tissue of the organism. The
bioavailability in eye topical administration is high, thus the
dose can be reduced, and the systemic toxicity can also be
reduced.
[0117] (d) The polypeptide of the present invention can be
synthesized via solid phase synthesis with high purity, high yield
and low cost.
[0118] (e) The polypeptide of the present invention is highly
stable.
[0119] Therefore, the polypeptide of the present invention can be
developed into a medicine for treating neovascular eye diseases and
related diseases associated with angiogenesis, such as tumor
angiogenesis, etc.
[0120] The invention is further illustrated by the following
examples. These examples are only intended to illustrate the
invention, but not to limit the scope of the invention. For the
experimental methods in the following examples the specific
conditions of which are not specifically indicated, they are
performed under routine conditions, e.g., those described by
Sambrook. et al., in Molecule Clone: A Laboratory Manual, New York:
Cold Spring Harbor Laboratory Press, 1989, or as instructed by the
manufacturers, unless otherwise specified.
Example 1
Synthesis, Seperation and Purification of Small Peptide ZY3
[0121] The polypeptides ZY3 represented by SEQ ID NO: 1 were
synthesized by using the commercially available SYMPHONY
polypeptide synthesizer (12-channel, Protein Technologies. LLC.,
U.S.). The processes were as follows:
[0122] The reagents were calculated and prepared according to the
software (Version. 201) of the polypeptide synthesizer.
2-Chlorotrityl Chloride Resin (Nankai Synthetic Technology Co.,
Ltd, Tianjin, China) was added into a reaction tubes, DMF (15 ml/g)
(Dikma) was added and the tube was oscillated for 30 min. Solvents
were suction filtered out through the sintered filter. 3-fold
excess mole of Fmoc-L-OH (small peptide ZY3) amino acids (Suzhou
Tianma Pharma Group Specialty Chemicals Co., Ltd.) was added and
then 10-fold excess mole of DIEA (Sinopharm Shanghai Chemical
Reagent Company) was added and finally, DMF was added for
dissolution. The mixture was oscillated for 30 min. DMF was removed
and 20% piperidine (Sinopharm Shanghai Chemical Reagent Company)
solution in DMF (15 ml/g) was added to react for 5 min. DMF was
removed and another 20% piperidine solution in DMF (15 ml/g) was
added to react for 15 min. Piperidine solution was suction
filtered. A dozen granules of resin were taken out and washed for
three times with ethanol. A droplet of ninhydrin solution, a
droplet of KCN solution, and a droplet of phenol solution were
added respectively. Then, the mixture was heated at 105.degree.
C.-110.degree. C. for 5 min and the change of color into dark blue
indicated the positive reaction. The resins were washed with DMF
(10 ml/g, twice), methanol (10 ml/g, twice), and DMF (10 ml/g,
twice). 3-fold excess of both protected amino acids (FOMC-Asp-OH)
and HBTU (Suzhou Tianma Pharma Group Specialty Chemicals Co., Ltd.)
were added respectively. They were dissolved with little DMF and
added into the reaction tube. Then 10-fold excess of NMM was
immediately added and mixture was reacted for 30 min. It was washed
with DMF (10 ml/g) once, methanol (10 ml/g) twice, and DMF (10
ml/g) twice. The above steps were repeated and the amino acids were
connected from right to left according to the sequence of small
peptides ZY3. After the connection of the last amino acid, the
deprotection was carried out and the resins were washed with DMF
(10 ml/g, twice), methanol (10 ml/g, twice), DMF (10 ml/g, twice)
and DCM (10 ml/g, twice) respectively. Then the resins were drained
for 10 min. The polypeptides were cleaved from the resins (cleavage
fluid (10/g): 94.5% TFA (J. T. Baker), 2.5% water, 2.5% EDT
(AlLDRICH), 1% TIS (AlLDRICH); cleavage time: 120 min). Residual
liquid containing protein was sufficiently dried with nitrogen
(Shanghai Biou Gas Industry Ltd.), washed for six times with
diethyl ether (Shanghai Shiyi Chemical Reagents Ltd.), and then
dried at room temperature.
[0123] Polypeptides were purified with HPLC (Models of SHIMADZU
HPLC device: preparation model/analytical model. Software:
Class-VP, Sevial System, manufacturer:
[0124] Shimadzu). The crude peptides were dissolved with pure water
or small amount of acetonitrile (Fisher) and small peptides ZY3
were purified under the following conditions:
[0125] Pump A: 0.1% trifluoroacetic acid+ultrapure water
[0126] Pump B: 0.1% trifluoroacetic acid+acetonitrile
[0127] Flow rate: 1.0 ml/min
[0128] Detection volume: 30 .mu.l
[0129] Wavelength: 220 nm
[0130] Detection column: Column:Venusi MRC-ODS C18 (30.times.250
mm)
[0131] The detection processes are shown in table 2
TABLE-US-00004 TABLE 2 Time (min) A (%) B (%) 0.5 90 10 30.0 20 80
30.1 stop
[0132] Finally, the purified solution was lyophilized to obtain
small peptides ZY3 with high purity (>95%).
Example 2
Identification and Storage of Small Peptides ZY3
[0133] A small amount of small peptides ZY3 was taken for purity
identification by HPLC analysis and molecular weight identification
by ESI-MS.
[0134] The results showed that the elution peak of ZY3 was at 12.8
min with the purity over 99% (FIG. 1).
[0135] Small peptide ZY3 has 27 amino acids in total with a
molecular weight of 3092.55. The small peptides in white powder
form were sealed, packaged, and stored at -20.degree. C.
Example 3
Effect of Small Peptides ZY3 on Proliferation Activity of
HUVECs
[0136] The MTS method was used as follows:
[0137] Primary Human Umbilical Vein Endothelial Cells (HUVECs)
(purchased from ScienCell Co.) were inoculated into a 96-well plate
with an inoculation concentration of 2.times.10.sup.4/ml. After
cells had adhered to the wall, serum-free culture medium ECM was
added and the cells were cultivated at 37.degree. C. for 24 hours.
Then the serum-free culture medium ECM as negative control, VEGF
(100 ng/ml) (purchased from Sigma Co.) as positive control, VEGF
(100 ng/well)+small peptide ZY3 in different concentrations as
treatment groups were added in each well. After a 24-hour
incubation, 20 .mu.l 1 MTS solution (purchased from Promega
Corporation) was added in each well. After incubation at 37.degree.
C. for 4 hours, the absorbance in each well was measured at 490 nm
by using microplate reader (Bio-Rad Co.). The proliferation
activity of cells was determined according to OD490. Finally,
SPSS11.0.1 was used for statistical analysis.
[0138] The results shown in FIG. 2 indicated that small peptide ZY3
significantly inhibited the proliferation of HUVECs and the
inhibitation was concentration-dependent. Compared with VEGF group,
in groups of VEGF+peptide ZY3 the proliferation of HUVECs was
significantly inhibited. *P<0.05, **P<0.01. The differences
are statistically significant.
Example 4
Effect of Small Peptide ZY3 on Lumen Formation of HUVECs
[0139] The Matrigel method was used as follows:
[0140] A 50 .mu.l/well Matrigel (purchased from BD Co.) was added
into a 96-well plate, and then the plate was incubated at
37.degree. C. for 30 min. Upon solidification, the primary HUVECs
were inoculated onto the surface of Matrigel with an inoculation
concentration of 8.times.10.sup.6/ml. Then the serum-free culture
medium ECM as negative control, VEGF (100 ng/ml) (purchased from
Sigma Co.) as positive control, VEGF (100 ng/well)+small peptide
ZY3 in different concentrations as treatment groups were added in
each well. The plate wad incubated at 37.degree. C. After treating
for 6 hours, photos were taken for cells in 3 randomized fields in
the plate under the microscope (.times.200 fold.), and the sum of
the maximum lumina diameter formed in the cells was calculated.
Finally, SPSS11.0.1 was used for statistical analysis.
[0141] The results shown in FIG. 3 indicated that small peptide ZY3
started to inhibit the lumen formation of HUVECs at the 6.sup.th
hour and the effect was concentration-dependent. FIG. 3a-3c showed
the lumen formation inhibition effects of small peptide ZY3 on
HUVECs. FIG. 3a is the VEGF group; FIG. 3b is the VEGF+ZY3 (160
.mu.M) group; FIG. 3c indicates that in groups of VEGF with Small
peptide ZY3 in different concentration, lumen formation of HUVECs
is significantly inhibited, compared with the VEGF
group.*P<0.05. The differences are statistically
significant.
Example 5
Detection of Inhibition Effect of Small Peptides ZY3 on
Angiogenesis in Chick Embryo Chorioallantoic Membrane
[0142] Model of chick embryo chorioallantoic membrane was used and
the methods were as follows:
[0143] Upon sterilization, the chick fertile eggs of 1-2 days old
(purchased from 36 Lianhuaqing Chicken Farm of Shanghai Xinghuo
Farm) were placed into a thermhygrostat (purchased from Shanghai
Boxun industrial Co., ltd. SPX-250C) (T=37.degree. C., Humidity
H=60-70%) to incubate for 5 days. Every day, the eggs were
overturned for one time at morning and night. Onto the filter paper
(Whatman quantitative filter papers, Sigma, ashless, Grade 42, Cat
No 1442-042, 42.5 mm .PHI..times.100 circles) containing cortisone
acetate (5 .mu.g/.mu.l, 5 .mu.l/piece), PBS (5 .mu.l/piece), low (2
.mu.g/.mu.l) or high (10 .mu.g/.mu.l) concentration of small
peptides ZY3 (5 .mu.l/piece) were dropped respectively. After
air-dried, the filter paper was placed between the major vessels on
chorioallantoic membrane of fertile eggs and the eggs were sealed.
The eggs were placed in the thermhygrostat (T=37.degree. C.,
Humidity H=60-70%) to incubate for another 2 days (24 hours a day)
without overturning. Then the chorioallantoic membrane was
thoroughly exposed and photographed (the range was within 5 mm
around the filter paper). The number of 3-5 subordinate
microvessels in the area within 2.5 mm around the filter paper was
counted. SPSS11.0.1 was used for statistical analysis.
[0144] The results shown in FIG. 4 indicated that compared with PBS
group, small peptide ZY3 exhibits significant inhabitation effects
on angiogenesis of chick embryo chorioallantoic membrane both in
low (10 .mu.g/piece) and high (50 .mu.g/piece) concentration. FIG.
4a-4c showed the counts of 3-5 subordinate microvessels in the area
within 2.5 mm around the filter paper. FIG. 4a is the PBS group;
FIG. 4b is the ZY3(10 .mu.l/piece) group; FIG. 4c is the ZY3(50
.mu.l/piece) group; FIG. 4d indicated that compared with the VEGF
group, in the groups of VEGF+small peptide ZY3 with different
concentrations, the amount of neovascularization in chick embryo
chorioallantoic membrane is significantly inhibited and the
inhibition is concentration-dependent. **P<0.01. The differences
are statistically significant.
Example 6
Detection of Inhibitation Effect of Small Peptide ZY3 on
Pathological Angiogenesis in the Mouse Cornea
[0145] Mice model of the corneal stroma micropocket was used and
the methods were as follows:
[0146] Male C57BL/6 mice (4-5 week old) were intraperitoneally
injected with 2% pentobarbital (about 0.1 ml/mouse) for anesthesia.
4% hydrochloric oxybuprocaine ophthalmic solution was locally
administrated. Under the stereo microscope, OT syringe needle and 2
ml syringe needle were used to perform a blunt dissection between
the layers of corneal stroma at 0.8-1 mm from corneoscleral limbus.
A pouch of about 0.6.times.0.8 mm was formed. Sustained-release
granules (mixture of 1:1 (v/v) of 12% PolyHAME: sucralfate) were
implanted respectively into the micropocket, including negative
control group (blank granule), positive control group (VEGF group:
320 ng/.mu.l, 160 ng/granule), and treatment group (VEGF+small
peptides ZY3 of low concentration (1 .mu.g/granule), or high
concentration (2 .mu.g/granule)). 5 days after operation, the
length of the longest new vessel (VL is the length of the longest
new blood vessel grown from limbus to the cornea), and the clock
hours of corneal neovascularization (CN represents the accumulative
clock hours of corneal neovascularization) were observed.
Neovascularization area were calculated as followed: Area
(mm.sup.2)=0.5*3.14*VL (mm)*CN*0.4 (mm) SPSS1L 0.1 was used for
statistical analysis.
[0147] The results shown in FIG. 5 indicated that compared with the
VEGF group, Small peptide ZY significantly inhibited the corneal
angiogenesis both in low concentration (0.5 .mu.g/granule) and high
concentration (2 mg/granule) (see the note). FIG. 5 showed the
effect of small peptide ZY3 on pathological angiogenesis in mouse
cornea, which demonstrated that small peptide ZY3 significantly
inhibits angiogenesis. FIG. 5a-5c show the neovascularization area
on mouse cornea. FIG. 5a is the VEGF group; FIG. 5b is the ZY3(0.5
.mu.l/granule) group; FIG. 5c is the ZY3(2 .mu.l 1/granule) group;
FIG. Sd indicated that compared with the VEGF group, in the groups
of VEGF+small peptide ZY3 with different concentrations, the
pathological angiogenesis in mouse cornea was significantly
inhibited. **P<0.01. The differences are statistically
significant.
Example 7
Preparation of Eyedrop
[0148] The following components were mixed via the conventional
techniques to obtain a 1% eyedrop, the formulation of which was
listed as follows:
TABLE-US-00005 ZY3 peptide (ZY3) 10 mg Hydroxylpropyl methyl
celloluse 0.03 g Sterile water q.s. to 10 ml
[0149] The osmotic pressure was adjusted to 300 Osm, and the pH was
adjusted to 6.8-7.1.
[0150] Five volunteers used the eyedrop for one week, three times
per day, and 1 drop/eye for each time. The results showed that the
eyedrop could inhibit ocular angiogenesis.
Example 8
Preparation and Activity of Derived Polypeptides
[0151] Derived Polypeptides were prepared as follows, and the
inhibition effect of each ZY3 derived polypeptides on proliferation
of Human Umbilical Vein Endothelial Cells HUVECs was determined
according to the methods in Example 3.
[0152] Derived Polypeptide 1: the sequence was the same as SEQ ID
NO.: 1 except that Val at position 4 was substituted by Ile.
[0153] Derived Polypeptide 2: the sequence was the same as SEQ ID
NO.: 1 except that Arg at position 12 was substituted by Pro.
[0154] Derived Polypeptide 3: the sequence was the same as SEQ ID
NO.: 1 except that Asp at position 15 was substituted by Glu.
[0155] Derived Polypeptide 4: the sequence was the same as SEQ ID
NO.: 1 except that Thr at position 23 was substituted by Arg.
[0156] Derived Polypeptide 5: the sequence was the same as SEQ ID
NO.: 1 except that Leu at position 8 was deleted.
[0157] The results indicated that in the treatment group (1
.mu.g/.mu.l) of the above derived polypeptides 1-5, the
proliferation of HUVEC was significantly inhibited.
[0158] All references mentioned in the present invention are
incorporated herein by reference, as each of them is individually
cited herein by reference. Further, it should be understood that,
after reading the above contents, the skilled person can make
various modifications or amendments to the present invention. All
these equivalents also fall into the scope defined by the pending
claims of the subject application.
Sequence CWU 1
1
2127PRTArtificial Sequenceangiogenesis-inhibiting peptide ZY3 1Thr
Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly Asp Arg 1 5 10
15 Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His 20 25
281DNAArtificial Sequenceencoding sequence for
angiogenesis-inhibiting peptide ZY3 2acggccaatg tcaccatgca
gctcctaaag atccgttctg gggaccggcc ctcctacgtg 60gagctgacgt tctctcagca
c 81
* * * * *